Drug-Inducible Gene Therapy Effectively Reduces Spontaneous Seizures in Kindled Rats but Creates Off-Target Side Effects in Inhibitory Neurons

Over a third of patients with temporal lobe epilepsy (TLE) are not effectively treated with current anti-seizure drugs, spurring the development of gene therapies. The injection of adeno-associated viral vectors (AAV) into the brain has been shown to be a safe and viable approach. However, to date, AAV expression of therapeutic genes has not been regulated. Moreover, a common property of antiepileptic drugs is a narrow therapeutic window between seizure control and side effects. Therefore, a long-term goal is to develop drug-inducible gene therapies that can be regulated by clinically relevant drugs. In this study, a first-generation doxycycline-regulated gene therapy that delivered an engineered version of the leak potassium channel Kcnk2 (TREK-M) was injected into the hippocampus of male rats. Rats were electrically stimulated until kindled. EEG was monitored 24/7. Electrical kindling revealed an important side effect, as even low expression of TREK M in the absence of doxycycline was sufficient to cause rats to develop spontaneous recurring seizures. Treating the epileptic rats with doxycycline successfully reduced spontaneous seizures. Localization studies of infected neurons suggest seizures were caused by expression in GABAergic inhibitory neurons. In contrast, doxycycline increased the expression of TREK-M in excitatory neurons, thereby reducing seizures through net inhibition of firing. These studies demonstrate that drug-inducible gene therapies are effective in reducing spontaneous seizures and highlight the importance of testing for side effects with pro-epileptic stressors such as electrical kindling. These studies also show the importance of evaluating the location and spread of AAV-based gene therapies in preclinical studies.

[1]  D. Kullmann,et al.  On-demand cell-autonomous gene therapy for brain circuit disorders , 2022, Science.

[2]  V. Solovyeva,et al.  Gene and Cell Therapy for Epilepsy: A Mini Review , 2022, Frontiers in Molecular Neuroscience.

[3]  K. Thomson,et al.  Spontaneous recurrent seizures in an intra-amygdala kainate microinjection model of temporal lobe epilepsy are differentially sensitive to antiseizure drugs , 2020, Experimental Neurology.

[4]  Gerlind Wallon,et al.  Hermann Bujard (1934 - 2020) - pioneering researcher and visionary science politician. , 2020, The EMBO journal.

[5]  John A. White,et al.  CaMKIIα-Positive Interneurons Identified via a microRNA-Based Viral Gene Targeting Strategy , 2020, The Journal of Neuroscience.

[6]  J. Kapur,et al.  Characterization of kindled VGAT‐Cre mice as a new animal model of temporal lobe epilepsy , 2020, Epilepsia.

[7]  M. Barker-Haliski,et al.  Validated animal models for antiseizure drug (ASD) discovery: Advantages and potential pitfalls in ASD screening , 2020, Neuropharmacology.

[8]  D. Kullmann,et al.  In vivo CRISPRa decreases seizures and rescues cognitive deficits in a rodent model of epilepsy , 2020, Brain : a journal of neurology.

[9]  R. Samulski,et al.  Engineering adeno-associated virus vectors for gene therapy , 2020, Nature Reviews Genetics.

[10]  D. Kullmann,et al.  Epilepsy Gene Therapy Using an Engineered Potassium Channel , 2019, The Journal of Neuroscience.

[11]  Esther Krook-Magnuson,et al.  Targeting the Mouse Ventral Hippocampus in the Intrahippocampal Kainic Acid Model of Temporal Lobe Epilepsy , 2018, eNeuro.

[12]  K. Chu,et al.  Clustering of spontaneous recurrent seizures separated by long seizure-free periods: An extended video-EEG monitoring study of a pilocarpine mouse model , 2018, PloS one.

[13]  J. Kapur,et al.  A novel therapeutic approach for treatment of catamenial epilepsy , 2018, Neurobiology of Disease.

[14]  J. Kapur,et al.  Electroencephalography and behavior patterns during experimental status epilepticus , 2018, Epilepsia.

[15]  Jing Liao,et al.  Identification and characterization of outcome measures reported in animal models of epilepsy: Protocol for a systematic review of the literature–A TASK2 report of the AES/ILAE Translational Task Force of the ILAE , 2017, Epilepsia.

[16]  A. Sharan,et al.  Prevalence and Incidence of Drug-Resistant Mesial Temporal Lobe Epilepsy in the United States. , 2017, World neurosurgery.

[17]  Mads Kærn,et al.  Improvement of the reverse tetracycline transactivator by single amino acid substitutions that reduce leaky target gene expression to undetectable levels , 2016, Scientific Reports.

[18]  F. D. Silva,et al.  Which insights have we gained from the kindling and post-status epilepticus models? , 2016, Journal of Neuroscience Methods.

[19]  Malisa Sarntinoranont,et al.  Convection-enhanced delivery to the central nervous system. , 2015, Journal of neurosurgery.

[20]  W. Stacey,et al.  On the nature of seizure dynamics. , 2014, Brain : a journal of neurology.

[21]  Jun-Hyeok Choi,et al.  Optimization of AAV expression cassettes to improve packaging capacity and transgene expression in neurons , 2014, Molecular Brain.

[22]  R. Stornetta,et al.  A potassium leak channel silences hyperactive neurons and ameliorates status epilepticus , 2014, Epilepsia.

[23]  J. González-Martínez,et al.  Reducing versus stopping antiepileptic medications after temporal lobe surgery , 2014, Annals of clinical and translational neurology.

[24]  N. Pillay,et al.  Patient perceptions and barriers to epilepsy surgery: Evaluation in a large health region , 2013, Epilepsy & Behavior.

[25]  Jeff W. Lichtman,et al.  NEW TOOLS FOR THE BRAINBOW TOOLBOX , 2013, Nature Methods.

[26]  Jian Ye,et al.  Primer-BLAST: A tool to design target-specific primers for polymerase chain reaction , 2012, BMC Bioinformatics.

[27]  I. Fried,et al.  Early surgical therapy for drug-resistant temporal lobe epilepsy: a randomized trial. , 2012, JAMA.

[28]  John S Duncan,et al.  The long-term outcome of adult epilepsy surgery, patterns of seizure remission, and relapse: a cohort study , 2011, The Lancet.

[29]  Linh Vong,et al.  Leptin Action on GABAergic Neurons Prevents Obesity and Reduces Inhibitory Tone to POMC Neurons , 2011, Neuron.

[30]  Jack J. Lin,et al.  The neurobiology of cognitive disorders in temporal lobe epilepsy , 2011, Nature Reviews Neurology.

[31]  M. Gossen,et al.  Improved Tet-responsive promoters with minimized background expression , 2010, BMC biotechnology.

[32]  C. Kilkenny,et al.  Guidelines for reporting experiments involving animals: the ARRIVE guidelines , 2010, British journal of pharmacology.

[33]  G. Mathern,et al.  Definition of drug resistant epilepsy: Consensus proposal by the ad hoc Task Force of the ILAE Commission on Therapeutic Strategies , 2009, Epilepsia.

[34]  R. Mandel,et al.  Tight Long-term dynamic doxycycline responsive nigrostriatal GDNF using a single rAAV vector. , 2009, Molecular therapy : the journal of the American Society of Gene Therapy.

[35]  Robert E Campbell,et al.  Exploration of new chromophore structures leads to the identification of improved blue fluorescent proteins. , 2007, Biochemistry.

[36]  A. Erisir,et al.  Selective loss of dentate hilar interneurons contributes to reduced synaptic inhibition of granule cells in an electrical stimulation‐based animal model of temporal lobe epilepsy , 2007, The Journal of comparative neurology.

[37]  D. Long,et al.  Models of Seizures and Epilepsy , 2006 .

[38]  Lippincott Williams Wilkins,et al.  Practice Parameter: Temporal lobe and localized neocortical resections for epilepsy: Report of the Quality Standards Subcommittee of the American Academy of Neurology, in Association with the American Epilepsy Society and the American Association of Neurological Surgeons , 2003, Neurology.

[39]  S. Kügler,et al.  Human synapsin 1 gene promoter confers highly neuron-specific long-term transgene expression from an adenoviral vector in the adult rat brain depending on the transduced area , 2003, Gene Therapy.

[40]  W T Blume,et al.  A randomized, controlled trial of surgery for temporal-lobe epilepsy. , 2001, The New England journal of medicine.

[41]  M. Lazdunski,et al.  A mammalian two pore domain mechano‐gated S‐like K+ channel , 1998, The EMBO journal.

[42]  R. Rhoads,et al.  Functional Characterization of the Internal Ribosome Entry Site of eIF4G mRNA* , 1998, The Journal of Biological Chemistry.

[43]  P. G. Reeves,et al.  Development and testing of the AIN-93 purified diets for rodents: results on growth, kidney calcification and bone mineralization in rats and mice. , 1993, The Journal of nutrition.

[44]  Edward H Bertram,et al.  Self-sustaining limbic status epilepticus induced by ‘continuous’ hippocampal stimulation: electrographic and behavioral characteristics , 1989, Epilepsy Research.

[45]  K. Berns,et al.  Nucleotide sequence of the inverted terminal repetition in adeno-associated virus DNA , 1980, Journal of virology.

[46]  R. Racine,et al.  Modification of seizure activity by electrical stimulation. II. Motor seizure. , 1972, Electroencephalography and clinical neurophysiology.

[47]  Edward M. Callaway,et al.  Short Promoters in Viral Vectors Drive Selective Expression in Mammalian Inhibitory Neurons, but do not Restrict Activity to Specific Inhibitory Cell-Types , 2009, Frontiers in neural circuits.

[48]  M. Wiznerowicz,et al.  A versatile tool for conditional gene expression and knockdown , 2006, Nature Methods.

[49]  J. Zentner,et al.  Surgical treatment of epilepsies. , 2002, Acta neurochirurgica. Supplement.

[50]  A. Wyler,et al.  Neuropsychological outcome following anterior temporal lobectomy in patients with and without the syndrome of mesial temporal lobe epilepsy. , 1998, Neuropsychology.

[51]  P. D. De Deyn,et al.  Epilepsy and the GABA-hypothesis a brief review and some examples. , 1990, Acta neurologica Belgica.

[52]  G. Paxinos,et al.  The Rat Brain in Stereotaxic Coordinates , 1983 .

[53]  R. Racine.  Modification of seizure activity by electrical stimulation: cortical areas. , 1975, Electroencephalography and clinical neurophysiology.